The Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has become widely adopted for DNA recognition, enabling applications such as genome-editing and recruitment of effector proteins to specific loci, to modulate transcription or enable direct imaging. Recently we showed that depending on specific modifications, Cas9 is able to bind specific mRNAs in living mammalian cells allowing tracking of their movement, or promotes degradation of target RNAs, opening up the potential for many RNA applications of Cas proteins. In this proposal, we seek to further expand the CRISPR/Cas toolbox for RNA modulation. To achieve our goal, the Corbett and Yeo labs will team up to use protein engineering, biochemistry, and cell biology techniques to (1) rationally design, then validate and optimize RNA-targeting activity of minimized Cas9 proteins from multiple species; (2) develop RNA-targeting Cas9 (RCas9) to visualize and track specific RNAs at single-molecule resolution in live cells; (3) develop RCas9 as a system for programmable editing and/or targeted destruction of repeat-containing mRNAs in human cells, and (4) adapt RCas9 for dynamic RNA control using chemically-inducible protein dimerization. Completion of the efforts outlined in this proposal will result in an expanded RNA-targeting Cas protein toolbox that will allow multiplex engineering of the transcriptome via direct editing of targeted RNA bases, programmable cleavage of disease- associated repeat-containing transcripts as well as other RNAs-of-choice with a universal RNA endonuclease, as well as a dynamically-controlled means to alter RNA metabolism and translation. These tools will provide a foundation for functional transcriptome engineering and in the future, enable development of therapeutics for myotonic dystrophy, C9ALS, Huntington?s disease and other conditions caused by repeat-containing RNAs.